This invention relates to single chain antibody molecules against the p53 gene product.
The following references are referenced in the specification and may be relevant to understanding the invention:
1. Gamble, J. and Milner, J. (1988). Evidence that immunological variants of p53 represent alternative protein conformations. Virology, 162, 452-458.
2. Harlow, E., Crawford, L. V, Pim, D. C. and Williamson, N. H. (1981). Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol., 39, 861-869.
3. Yewdell, J. W., Gannon, J. V. and Lane, D. P. (1986). Monoclonal antibody analysis of p53 expression in normal and transformed cells. J. Virol. 59, 444-452.
4. Milner, J., Cook, A. and Sheldon, M. (1987). A new anti-p53 monoclonal antibody, previously reported to be directed against the large T antigen of simian virus-40. Oncogene, 1, 453-455.
5. Gannon, J. V., Greaves, R., Iggo, R. and Lane, D. P. (1990). Activating mutations in p53 produce common conformation effects. A monoclonal antibody specific for the mutant form. EMBO J. 9, 1595-1602.
6. Marasco, W. A. (1995) Intracellular antibodies (intrabodies) as research reagents and therapeutic molecules for gene therapy. Immunotechnology, 1, 1-19.
7. Stephen, C. W and Lane, D. P. (1992). Mutant conformation of p53 precise epitope mapping using a filamentous phage epitope library. J. Mol. Biol. 225, 577-583.
8. Jannot, C. B. and Hynes, N. E. (1997). Biochem. Biophys. Res. Commun., 230, 242-246.
9. Cohen, P. A., Mani, J-C. and Lane, D. P. (1998). Characterization of a new intrabody directed against the N-terminal region of human p53. Oncogene, 17, 2445-2456.
The p53 gene encodes a protein which is 53 kD in size (hence the name). The p53 protein is situated in the nexus of intermingled pathways controlling cell proliferation, cell survival and differentiation. It can sense and integrate various external and internal stimuli, such as DNA damage, hypoxia, oxidative stress, deregulated oncogene expression and ribonucleotide depletion. In response to these stimuli, it may trigger cell cycle arrest, apoptosis, senescence, differentiation or antiangiogenesis. It appears that the p53 protein is involved in regulation of critical growth controlling checkpoints. It is capable of exercising a tumor suppressor function by preventing cells, that are in unfavorable environmental conditions or are carrying damaged DNA, from entering a cell cycle.
Mutations within the p53 gene have been found in more than 50% of all human cancers, rendering it the most frequently mutated single gene in human cancer known so far. Most of the mutations in the p53 gene discovered in cancers are of a missense type. They cause not just abrogation of the tumor suppressor function of wild-type p53, but often actively contribute to the tumor transformation function of mutant p53. Inactivation of p53 is one of the most common molecular events in cancer development.
As mentioned above, the p53 protein, which is both a regulator for cell proliferation and a suppressor of tumor development, can prevent the development of cancer by blocking the division of cells which have sustained DNA damage, or by triggering apoptosis. It has been proposed that the effect of mutant p53 on tumor progression is due to a dominant negative interaction of the wild type and mutant proteins. Thus, wild-type and mutant p53 proteins are respectively capable of contrasting suppressor and promoter effects on tumor development.
The ability of p53 to act both as a suppressor and a promoter of tumor development may reflect the ability of the protein to adopt different conformations. Mitogenic stimulation of primary T cells induces a change in the immunoreactivity of p53 and this may be due to a change in the tertiary structure of the p53 protein. To ascertain whether the xe2x80x98mutantxe2x80x99 conformation of wild-type p53 has physiological relevance, attempts have been made to associate it with a biochemical activity. Conformation-specific monoclonal antibodies, previously shown to discriminate between wild-type and mutant p53 proteins, have been used to demonstrate structural changes in wild-type p53 following sequence-specific binding to DNA (1). These studies suggested that wild-type p53 can physiologically adopt distinct conformations, which determine its DNA binding activity. Mutations that render p53 oncogenic may lock p53 into one of the few conformational states it physiologically adopts, rather than distort its tertiary structure.
Two important cellular factors appear necessary for this conversion: (1) binding of an xe2x80x98activatingxe2x80x99 polypeptide that causes neutralization of the C-terminal negative regulatory domain, and (2) a highly reduced environment which can maintain p53 in an activated state. Given the clear association between the DNA binding activity and tumor suppressor functions of p53, these results imply that in many tumor cells there are high levels of mutant p53 that can potentially be activated to restore significant wild type function.
A variety of monoclonal antibodies have been prepared against various epitopes of p53.
A panel of anti-p53 mouse monoclonal antibodies has been used to characterize the immunoreactivities of the native p53-Ala35 and the mutant p53-Val 35 translated under various conditions. Two monoclonal antibodies, PAb421 and PAb248, were able to recognize discrete denaturation-stable epitopes on the p53 polypeptide (2,3). These antibodies immunoprecipitated both p53-Ala35 and p53-Val 35 translated at 30xc2x0 C. and 37xc2x0 C., and were also able to immunoprecipitate p53 from a variety of murine cell lines (4).
Three additional monoclonal antibodies, Pab246, Pab1620 and Pab240, were found to detect conformational changes in the p53 protein (5). Molecules in the mutant conformation are distinguished from wild-type molecules, inter alia, by the appearance of a new, normally cryptic epitope recognized by Pab240. This epitope was localized to residues 213 to 217 of the p53 protein and has the sequence RHSVV, SEQ ID NO: 9, preceded by F in human and mouse p53(7). More than 90% of mutations found in p53 produce a conformational change in the p53 protein which results in the exposure of this epitope, which is otherwise hidden in the hydrophobic core of the molecule. This epitope will be referred to hereinafter as the common mutant epitope of p53.
Recent advances in antibody engineering have allowed the genes encoding antibodies to be manipulated, so that antigen binding molecules can be expressed within mammalian cells in a controlled way (6). Application of gene technologies to antibody engineering has enabled the synthesis of single-chain fragment variable (scFv) antibodies that combine within a single polypeptide chain the light and heavy chain variable domains of an antibody molecule covalently joined by a predesigned peptide linker. The resultant scFv gene can be expressed in bacterial expression systems such as E. coli. Bundled in the xe2x80x9cgene display packagexe2x80x9d single-chain antibodies displayed at the surface of filamentous phages of the M13 family provided the possibility to create antibody libraries both from various living sources and products of diversification of a single scFv molecule. Antibodies with the desired specificity can be isolated from such libraries employing effective selection techniques (biopanning) in which the antigen is immobilized on a solid support.
The ability to create scFv antibodies, when combined with their stable expression in precise intracellular locations in mammalian cells, has enabled the creation of a powerful new family of antibody molecules for basic research or gene therapy. These intracellular antibodies (intrabodies) can be used to modulate cellular physiology and metabolism through a variety of mechanisms, including the blocking, stabilizing or mimicking of protein-protein interactions, by altering enzyme f unction, or by diverting proteins from their usual intracellular compartments. Intrabodies can be directed to the relevant cellular compartments by modifying the genes that encode them to specify N- or C-terminal polypeptide extensions for providing intracellular-trafficking signals. This approach has been described for a number of different antigens, including several HIV proteins.
Several scFv antibodies against p53 have been previously described. The scFv-421 antibody recognizes a C-terminal epitope of the protein (8). When expressed in vitro in the cytoplasm or in the nucleus of COS-1 cells, it was found to be non-functional and prone to rapid degradation. An important determinant of correct antibody folding is the formation of intra-chain disulfide bonds in the variable regions; possibly the reducing environment of the cytosol may lead to a decrease in the stability of the scFv (9). Nevertheless, some scFv antibodies have been expressed in the cytoplasm and shown to have biological effects, indicating that other features, such as the primary sequence of the antibody and/or its specific cellular location may be important for their proper function.
The scFv DO-1 antibody was found to recognize an N-terminal epitope of human p53 (9). The DO-1 scFv was targeted to the cytoplasm and to the nucleus of mammalian cells. Interestingly, insertion of the CK domain into scFv to create a scFvCK fusion protein, led to a dramatic increase in the level of intracellular expression. However, in other studies in which CK fusions to scFv were made, the effects on stability and expression were much less marked. It is clear that each scFv is a particular and individual case (9).
It is an object of the present invention to provide a single chain antibody which recognizes an epitope exposed on mutant, but not on wild-type, p53.
Thus, in a first aspect the present invention provides a DNA molecule encoding a single chain antibody (scFv) which specifically recognizes the common mutant epitope in mutant p53 but not in wild type p53.
In a preferred embodiment, the scFv is ME1.
In a most preferred embodiment, the DNA molecule of the invention comprises SEQ. ID. NO. 1.
In the present specification, the normally cryptic, mutant p53 epitope motif as described above (FRHSVV, SEQ ID NO:8) which is recognized by the Pab240 antibody (7) is termed the xe2x80x9ccommon mutant epitopexe2x80x9d of mutant p53. This epitope differs from the p53 epitopes recognized by, the previously disclosed scFv antibodies mentioned above.
In order to realize the object of the invention, the gene segments encoding variable parts of the antibody heavy and light chains were amplified by PCR from the spleen of the hyperimmunized mouse, and a library of the antibody genes was obtained. When the genes isolated from the antibody gene library were assembled in the scFv DNA, expressed as phage antibodies and subjected to panning, the single-chain scFv ME1 that was isolated possessed a significant affinity (10xe2x88x927 M) towards mutant p53 and was successfully expressed as a soluble antibody, separate from the phage fusion.
Such libraries usually contain a large number of different genes encoding the antibodies specific to the chosen antigen, in contrast to a single pair (VH and VL) of antibody genes encoding a single antibody as present in hybridoma cells. This issue has a special importance for the amplification of mouse antibody genes because the sequencing of their repertoire has not yet been completed, and thus it is still not possible to design a primer set covering all existent antibody gene variants. Also, some single-chain antibody genes are difficult to express in bacterial cells for various reasons, among which are their toxicity for the host, low conformational stability and rapid proteolytic degradation. Thus, it appears that a much improved starting point for scFv construction is selecting from the collection of variants of the VH and VL domains present in the immunized host, than from a hybridoma cell line.
One or more nucleotides of the DNA molecule of the invention may be modified without affecting the ability of the antibody, encoded by the modified DNA molecule, to specifically recognize the common mutant epitope in mutant p53 but not in wild type p53. Such modifications are well known to the skilled man of the art, and include (1) substitutions, e.g. based on the degeneracy of the genetic code, and (2) insertions or deletions of nucleotide base triplets resulting in insertions to or deletions from the amino acid sequence of the scFv at non-essential locations. The modifications may be carried out by various techniques such as site-directed mutagenesis.
Codons preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al. Nuc Acids Res., 17:477-508, (1989)) can be selected, for example, to increase the rate of variant product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence. In a further preferred embodiment of this aspect of the invention, the DNA molecule comprises SEQ. ID. NO: 3, which has been modified for eukaryotic expression.
The DNA sequence of the present invention can be engineered in order to alter a scFv product coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the product. For example, alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.
The invention also relates to a vector, such as a plasmid or viral vector, into which the DNA molecule of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. A preferred vector is the expression vector pIRES-EGFP-ME1.
The present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention which may be for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the variant nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. A preferred host cell is a mammallian host cell containing the pIRES-EGFP-ME1 vector.
In a second aspect, the invention provides a scFv molecule which specifically recognizes the common mutant epitope in mutant p53 but not in wild type p53.
In a preferred embodiment, the scFv is ME1.
In a most preferred embodiment, the scFv comprises the amino acid sequence SEQ. ID. NO. 2. In another preferred embodiment, the scFv comprises SEQ. ID. NO: 4.
One or more amino acids of the scFv of the invention may be modified without affecting the ability of the antibody to specifically recognize the common mutant epitope in mutant p53 but not in wild type p53. Such modifications are well known to the skilled man of the art, and include (1) substitutions, e.g. substituting hydrophilic or hydrophobic amino acids with other hydrophilic or hydrophobic amino acids, respectively, by site directed mutagenesis, (2) insertions or deletions of amino acids at non-essential locations, and (3) chemical modifications.
Thus, the invention also includes a polypeptide comprising a polypeptide sequence having at least a 95% sequence identity, and more preferably at least a 99% sequence identity, to SEQ. ID. NO. 2, wherein said polypeptide sequence specifically recognizes the common mutant epitope in mutant p53 but not in wild type p53.
The modifications of the DNA molecule or of the scFv molecule may be directed towards conferring upon the scFv polypeptide various characteristics such as (1) increased specificity for the mutant p53 molecule as compared to the wild type p53 molecule, (2) higher affinity for the mutant p53 antigen, (3) increased stability and resistance to proteolysis, (4) enhanced expression and solubility of the scFv antibody in vitro and in vivo, and (5) preferred targeting of the scFv antibody to sub-cellular sites by incorporation into the scFv antibody of, for example, ER and nuclear targeting peptide sequences, so as to generate preferred embodiments of the invention for pharmacological and pharmaceutical applications. An example of a domain of the scFv which may be modified is the CDR domain. These alterations can be achieved not only by the introduction of nucleotide changes in the cloned scFv antibody gene encoding the polypetide using commonly known methods of chemical and enzymatic mutagenesis, such as oligonucleotide-directed mutagenesis and PCR-based mutagenesis (see Current Protocol in Molecular Biology, John Wiley and Sons,Inc., 1997, Volume 1, section 8), but also by chemical changes in the amino acid sequence of the scFv, such as glycosylation and by the creation of polyvalent scFv antibodies (see Smythe J. A. et al., (1994) Protein Engineering 7:145-147).
The scFv antibody of the invention has distinct advantages over the existing monoclonal antibodies. Thus, the modifications outlined above can readily be made with the scFv antibody but not with the monoclonal antibody. The smaller size of the scFv is also an advantage in intracellular applications.
In a third aspect, the invention provides a pharmaceutical composition comprising either a DNA molecule, a vector, or an antibody molecule according to the invention, and a pharmaceutically acceptable excipient.
In a fourth aspect, the invention provides a method for treating a patient suffering from a disease whose etiology is related to a mutation in the p53 gene comprising administrating to said patient a pharmaceutical composition according to the invention.
The scFv of the invention may be useful in the treatment of a disease whose etiology is related to a mutation in the p53 gene, and in particular, in the treatment of cancer.
A novel and promising approach in the gene therapy of tumors lies in the intracellular expression of antibodies that are capable of inactivating certain oncogene products, or by targeting their degradation. Because mutant p53 exerts distinct oncogenic properties and appears in the cytosol of a wide range of tumors, an intracellularly expressed single-chain antibody (intrabody) directed against this protein may serve as a xe2x80x9cbroad spectrumxe2x80x9d agent for tumor therapy. To adapt the ME1 scFv for conditions of intracytosolic mammalian expression, several modifications were introduced in the scFv DNA, as will be described more particularly below.
The scFv ME1 of the invention may serve as a powerful auxiliary agent capable of significantly enhancing the specificity and effectiveness of the two major existent anti-cancer gene therapies.
One of these strategies employs an overexpression of the wild-type p53 protein in cancer cells. In spite of the promising results obtained from several clinical trials utilizing this technique, it was recently found that cancer cells containing a mutant form of p53 are largely recalcitrant to this treatment. Expression of the scFv ME1 molecule as an intrabody fused to the F-box domain responsible for the targeting of the cell proteins to the degradation cascade may be capable of significantly reducing the level of mutant p53 in the cell, thereby broadening the range of possible tumor targets for the original therapy.
Another emerging anti-cancer gene therapy employs a single-chain antibody directed to a p53 protein epitope which is present both in wild-type and mutant p53 molecules. It forms a part of the synthetic transcription factor containing also the bacterial tetracycline repressor as a DNA binding domain. The strategy is based on the fact that the mutant form of p53 antibody serve as a tether bringing together a transactivation function provided by p53 and the DNA binding activity from the tetracycline receptor. The resultant complex can activate the transcription of the protein toxin put under control of the promoter containing tetracycline-operator sequences. The major drawback of this strategy is the indiscriminate nature of the antibody employed which causes an activation of toxin expression in a cell containing any form of p53 protein. As a consequence, only the local administration of this treatment can be considered as safe. The substitution of the original antibody by the scFv ME1 specific to the mutant form of p53 may restrict the therapeutic effect to cancer cells only, allowing a systemic application of this therapy.
In addition to its the clinical importance, the scFv ME1 antibody can serve as a valuable research and diagnostic tool, allowing specific tagging of mutant p53 molecules inside the cell. Mutation of the p53 gene results in stabilization of the protein and a subsequent increase in intracellular protein sufficient to be detectable by immunohistochemistry. The high specificity of the scFv of the invention towards a peptide epitope, which appears only in mutant variants of p53, the lack of the Fc portion which binds specifically to the antigen, and the high permeability of these small antibodies into cells, make the antibody of the invention a suitable probe for immunodiagnostic clinical detection of mutant p53 in tissues, using 20 conventional immunohistochemistry techniques. An immunodiagnostic kit could therefore be prepared comprising the scFv of the invention. Such kits using other antibodies for detecting other antigens are well known in the art.